Hematopoietic stem cells are a population of cells capable of both self renewal and of differentiation into a variety of hematopoietic lineages. (S. Huang and L. Terstappen, Nature, 360:745-749 (1992)). The definition of the hematopoietic stem cell is functional and based upon the ability of transplanted cells to repopulate the hematopoietic system of a recipient who has undergone myeloablative treatment. The prevailing view is that stem cells represent perhaps 0.01 percent of bone marrow cells, that they can self-renew and that they can be assayed by their ability to regenerate the bone marrow and to give rise to long-term lympho- and myelopoiesis. (M. Dexter and T. Allen, Nature, 360:709-710 (1992)).
The differentiation of hematopoietic cells from primitive multipotent progenitors to mature blood elements involves a series of lineage commitment steps accompanied by the acquisition of specific phenotypic characteristics. Cells gain or lose antigenic features and responsiveness to specific cytokines based on their lineage and stage of differentiation.
The efficient isolation of hematopoietic stem cells would enhance both the investigation of the processes of lineage commitment and self-renewal and the clinical strategies of bone marrow transplantation and hematopoietic cell gene therapy. However, identification of such primitive cells is challenging because of their apparent low frequency and their lack of identifiable morphologic features.
Several methods of enrichment have been developed based on immunologic and certain functional properties of primitive hematopoietic cells. For example, antibodies have been generated which recognize cell surface antigens associated with hematopoietic cells at various stages of lineage differentiation. Immunophenotypes which are enriched for fractions of human bone marrow cells with more primitive phenotypes are CD34+, CD33-, CD38-, HLA DR-, Thy-1.sup.lo and negative staining for lineage specific antigens (W. Craig, et al., J. Exp. Med. 177:1331 (1993); C. I. Civin et al., J. Immunol. 133:157 (1994); C. I. Civin, et al., Exp. Hematol. 15:10 (1987); L. W. M. M. Terstappen, et al., Blood 77:1218 (1991)). In addition, it has been recognized that low intensity of staining with the dye, Rhodamine.sub.123 (Rho), is associated with a fraction of bone marrow cells enriched for repopulating stem cells (R. E. Ploemacher, N. H. C. Brons, Exp. Hematol. 16:903 (1988); N. S. Wolf, et al., Exp. Hematol. 21:614 (1993); G. J. Spangrude, G. R. Johnson, Proc. Natl. Acad. Sci. U.S.A. 87:7433 (1990)).
These methods, however, depend upon technically demanding cell sorting which involves considerable mechanical manipulation of the cells. Further, antibody binding to cell surface structures may theoretically be capable of inducing perturbations of the cells' physiology.
Enrichment methods have been developed which exploit the observations that soy-bean agglutinin binding is uncommon in primitive bone marrow cells (Y. Reisner, et al., Blood 59:360 (1992); W. Ebell, et al., Blood 65:1105 (1985)) and that stem cells are relatively resistant to the toxic effects of the alkylating agent, 4-hydroxycyclophosphamide (4-HC) (M. Y. Gordon, et al., Leukemia Res. 9:1017 (1985); C. Smith, et al., Blood 77:2122 (1991)). The anti-metabolite, 5-fluorouracil (5-FU), when added to short-term bone marrow cultures was noted to enrich for early hematopoietic cells, presumably due to the relative quiescence of more primitive cells (C. Lemer, D. Harrison, Exp. Hematol. 18:114 (1990); M. F. Stewart et al., Blood 81, 2283 (1993)). However, these methods also suffer from limited selectivity, yielding relatively heterogeneous populations of precursor cells. Thus, a need still exists for an efficient method of isolating hematopoietic stem cells.